1. Field of the Invention
This invention relates primarily to functional DNA polynucleotides that exhibit allosteric properties, and to catalytic RNA and DNA polynucleotides that have catalytic properties with rates that can be controlled by a chemical effector, a physical signal, or combinations thereof. Bioreactive allosteric polynucleotides of the invention are useful in a variety of applications, particularly as biosensors.
Biosensors are widely used in medicine, veterinary medicine, industry, and environmental science, especially for diagnostic purposes. Biosensors are typically composed of a biological compound (sensor material) that is coupled to a transducer, in order to produce a quantitative readout of the agent or conditions under analysis. Usually, the biological component of the biosensor is a macromolecule, often subject to a conformational change upon recognition and binding of its corresponding ligand. In nature, this effect may immediately initiate a signal process (e.g., ion channel function in nerve cells). Included in the group of xe2x80x98affinity sensorsxe2x80x99 are lectins, antibodies, receptors, and oligonucleotides. In biosensors, ligand binding to the affinity sensor is detected by optoelectronic devices, potentiometric electrodes, field effect transistors (FETs), or the like.
Alternatively, the specificity and catalytic power of proteins have been harnessed to create biosensors that operate via enzyme function. Likewise, proteins have been used as immobilized catalysts for various industrial applications. The catalytic activity of purified enzymes or even whole organelles, microorganisms or tissues can be monitored by potentiometric or amperometric electrodes, FETs, or thermistors. The majority of biosensors that are commercially available are based on enzymes, of which the oxidoreductases and lyases are of great interest. It is nearly exclusively the reactants of the reactions catalyzed by these enzymes for which transducers are available. These transducers include potentiometric electrodes, FETs, pH- and O2-sensitive probes, and amperometric electrodes for H2O2 and redox mediators. For example, the oxidoreductases, a group of enzymes that catalyze the transfer of redox equivalents, can be monitored by detectors that are sensitive to H2O2 or O2 concentrations.
Enzymes are well-suited for application in sensing devices. The binding constants for many enzymes and receptors can be extremely low (e.g., avidin; Kd=10xe2x88x9215 M) and the catalytic rates are on the order of a few thousand per second, but can reach 600,000 secxe2x88x921 (carboanhydrase) (45). Enzymes can be monitored as biosensors via their ability to convert substrate to product, and also be the ability of certain analytes to act as inhibitors of catalytic function.
Organic chemistry and biochemistry have reached a state of proficiency where new molecules can be made to simulate the function of protein receptors and enzymes. Macrocyclic rings, polymers for imprinting, and self-assembling monolayers are now being intensively investigated for their potential application in biosensors. In addition, the immune system of animals can be harnessed to create new ligand-binding proteins that can act as artificial biorecognition systems. Antibodies that have been made to bind transition-state analogues can also catalyze chemical reactions, thereby functioning as novel xe2x80x98artificial enzymesxe2x80x99 (36). The latter examples are an important route to the creation of biosensors that can be used to detect non-natural compounds, or that function under non-physiologic conditions.
2. Description of Related Art
In nature, RNA not only serves as components of the information transfer process, but also performs tasks that are typically accomplished by proteins, including molecular recognition and catalysis. A seemingly endless variety of aptamers, and even DNA aptamers can be created in vitro that bind various ligands with great affinity and specificity (17). Nucleic acids likely have an extensive and as yet untapped ability adopt specific conformations that can bind ligands and also to catalyze chemical transformations (16). The engineering of new RNA and DNA receptors and catalysts is primarily achieved via in vitro selection, a method by which trillions of different oligonucleotide sequences are screened for molecules that display the desired function. This method consists of repeated rounds of selection and amplification in a manner that simulates Darwinian evolution, but with molecules and not with living organisms (4). One drawback to the use of existing enzymes as biosensors is that one is limited to developing a sensor based on the properties of existing enzymes or receptors. A significant advantage can be gained if one could xe2x80x98tailor-makexe2x80x99 the sensor for a particular application. It would be desirable to employ nucleic acids to create entirely new biosensors that have properties and specificities that span beyond the range of capabilities of current biosensors.
In vitro selection has been the main vehicle for new ribozyme discoveries in recent years. The catalytic repertoire of ribozymes includes RNA and DNA phosphoester hydrolysis and transesterification, RNA ligation, RNA phosphorylation, alkylation, amide and ester bond formation, and amide cleavage reactions. Recent evidence has shown that biocatalysis is not solely the realm of RNA and proteins. DNA has been shown to form catalytic structures that efficiently cleave RNA (5,7), that ligate DNA (10), and that catalyze the metallation of porphyrin rings (24). As described herein, self-cleaving DNAs have been isolated from a random-sequence pool of molecules that operate via a redox mechanism, making possible the use of an artificial DNA enzyme in place of oxidoreductase enzymes in biosensors. In addition, these DNA enzymes or xe2x80x98deoxyribozymesxe2x80x99 are considerably more stable that either RNA or protein enzymesxe2x80x94an attractive feature for the sensor component of a biosensor device.
It is an object of the invention to provide examples of RNA and DNA sensing elements for use in biosensors, including polynucleotides attached to a solid support. Both RNA and DNA can be designed to bind a variety of ligands with considerable specificity and affinity. In addition, both RNA and DNA can be made to catalyze chemical trans formations under user-defined conditions. A combination of rational design and combinatorial methods has been used to create prototype biosensors based on RNA and DNA.
These and other objects of the invention are accomplished by the present invention, which provides purified functional DNA polynucleotides that exhibit allosteric properties that modify a function or configuration of the polynucleotide with a chemical effector, a physical signal, or combinations thereof. The invention further provides purified functional polynucleotides having catalytic properties with rates that can be controlled by a chemical effector, a physical signal, or combinations thereof. Some embodiments are enzymes exhibiting allosteric properties that modify the rate of catalysis of the enzyme. In addition, the invention encompasses biosensors comprising bioreactive allosteric polynucleotides described herein.
Examples of chemical effectors include, but are not limited to, organic compounds such as amino acids, amino acid derivatives, peptides, nucleosides, nucleosides, nucleotides, steroids, and mixtures of organic compounds and metal ions. In some embodiments, the effectors are microbial or cellular metabolites or components of bodily fluids such as blood and urine obtained from biological samples. In other embodiments, the effectors are pharmaceuticals, pesticides, herbicides, and food toxins. Physical signals include, but are not limited to, radiation and temperature changes.
The invention also provides methods for determining the presence or absence of compounds, or compound concentrations in biological, industrial, and environmental samples, and physical changes in such samples using bioreactive allosteric polynucleotides of the invention and biosensors incorporating them.